Isolation, identification and synthesis of an endogenous arachidonic amide that inhibits calcium channel antagonist 1,4-dihydropyridine binding

This study was part of a broad search for endogenous regulators of L-type calcium channels. The screening for active fractions was done by measuring inhibition [³H]1,4-dihydropyridine (DHP) binding to rat cardiac and cortex membranes. An inhibitory fraction, termed lyophilized brain hexane-extractable inhibitor (LBHI), was isolated from hexane extracts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. ¹³C nuclear magnetic resonance (NMR), ¹H coherence spectroscopy (COSY) NMR and fast atom bombardment (FAB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hydroxyethylamide. Synthesis of this substance and subsequent high performance liquid chromatography (HPLC) and NMR analysis confirmed this structure. Synthetic LBHI (SLBHI) inhibited [³H]DHP binding to rat cortex membranes with an IC₅₀ value of 15 μM and a Hill coefficient of 2. Saturation analysis in the presence of SLBHI showed a change in K_D (equilibrium dissociation constant), but not maximal binding capacity (B_max). SLBHI produced an increased dissociation rate, which, along with the Hill slope of > 1, suggested a non-competitive interacton with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the DHP calcium antagonist binding site.     

IMMUNOCYTOCHEMICAL CHARACTERIZATION OF THE INTRAPYRENOID THYLAKOIDS OF CRYPTOMONADS1

Thylakoid lamellae extend into the pyrenoids of only two genera of cryptomonad algae, Chroomonas and Hemiselmis, We used immunoelectron microscopy to assess the photosynthetic competency of cryptomonad intrapyrenoid thylakoids. Intrapyrenoid thylakoids possess phycobiliproteins and the chlorophyll a/c₂ light-harvesting complex, both of which are associated with photosystem (PS) II in a light-harvesting capacity. In addition, thylakoids that extend into the pyrenoid of Hemiselmis brunnescens were immunolabelled by anti-PSI. These results indicate that cryptomonad intrapyrenoid thylakoids likely function in a manner analogous to thylakoids of the chloroplast stroma. Moreover, our observation that the Calvin cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is pyrenoid-localized in these two cryptophytes indicates that the processes of photosynthetic O₂-evolution and ribulose 1,5-bisphosphate (RuBP) carboxylation/oxygenation are not spatially separated in these algae.     

Stoichiometric relations in an ant-treehopper mutualism

Carbon : nitrogen : phosphorus (C : N : P) stoichiometry can underlie physiological and life history characteristics that shape ecological interactions. Despite its potential importance, there is much to learn about the causes and consequences of stoichiometric variation in terrestrial consumers. Here we show that treehoppers (Publilia modesta) tended by ants (Formica obscuripes) contained lower N concentrations than treehoppers on plants from which ants were excluded. Ant presence also affected nutrient concentrations in host plants: on plants with ants, leaves contained uniformly low concentrations of N; on plants without ants, N concentrations were low only in the few leaves fed upon by treehoppers at the time of collection. We suggest treehopper feeding reduces leaf nutrient levels and ants positively affect treehopper abundance, producing a top–down effect on plant quality. Determining the causes of these stoichiometric changes should help elucidate factors guiding the dynamics of conditional mutualisms between ants and homopterans.     

Independent cerebral vasoconstrictive effects of hyperoxia and accompanying arterial hypocapnia at 1 ATA.

Breathing 100% O2 at 1 atmosphere absolute (ATA) is known to be associated with a decrease in cerebral blood flow (CBF). It is also accompanied by a fall in arterial Pco2 leading to uncertainty as to whether the cerebral vasoconstriction is totally or only in part caused by arterial hypocapnia. We tested the hypothesis that the increase in arterial Po2 while O2 was breathed at 1.0 ATA decreases CBF independently of a concurrent fall in arterial Pco2. CBF was measured in seven healthy men aged 21-62 yr by using noninvasive continuous arterial spin-labeled-perfusion MRI. The tracer in this technique, magnetically labeled protons in blood, has a half-life of seconds, allowing repetitive measurements over short time frames without contamination. CBF and arterial blood gases were measured while breathing air, 100% O2, and 4 and 6% CO2 in air and O2 backgrounds. Arterial Po2 increased from 91.7 +/- 6.8 Torr in air to 576.7 +/- 18.9 Torr in O2. Arterial Pco2 fell from 43.3 +/- 1.8 Torr in air to 40.2 +/- 3.3 Torr in O2. CBF-arterial Pco2 response curves for the air and hyperoxic runs were nearly parallel and separated by a distance representing a 28.7-32.6% decrement in CBF. Regression analysis confirmed the independent cerebral vasoconstrictive effect of increased arterial Po2. The present results also demonstrate that the magnitude of this effect at 1.0 ATA is greater than previously measured.     

Bacteria Associated with Cysts of the Soybean Cyst Nematode (Heterodera glycines)

The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world. The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops. To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means. Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst. Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 ± 0.5) × 10⁵ bacteria (mean ± standard deviation) with various cellular morphologies. Filamentous fungi were also observed. Live-dead staining indicated that the majority of cyst bacteria were viable. The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature. Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition. Bands shared by all eight samples included the actinobacterium genera Actinomadura and Streptomyces. A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium. These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence. Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp. This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.     

Characterisation of Antibodies Specific for Chick Brain Neural Cell Adhesion Molecules Which Cause Amnesia for a Passive Avoidance Task

Abstract: Antisera were prepared against six postsynaptic density glycoprotein fractions (150–180, 62–80, 50, 41, 33, and 28 kDa) that show enhanced fucosylation during memory formation after training day-old chicks in a one-trial passive avoidance task. Each antiserum was tested for its possible effect on memory retention. Bilateral intracranial injections of two of the antisera, R-1 and R-6, or their IgGs (IgG-1 and IgG-6), resulted in amnesia for the passive avoidance task when chicks were tested 24 h later. IgG-1 and IgG-6 antibodies were amnestic only when injected 5.5 h after training, and had no effect when injections were made 30 min before training, thus resembling an effect previously observed with polyclonal or monoclonal anti-N-CAM antibodies. IgG-1 and IgG-6 antibodies were found to be specific for protein epitopes of glycoproteins that contain a high amount of N-linked mannose and fucose, and a very low amount of polysialic acid and O-linked galactose. Absorption of IgG-6 antibodies with neural cell adhesion molecule (N-CAM) isolated from synaptic plasma membranes derived from day-old chick brain resulted in loss of amnestic effect. As we have previously shown that long-term memory for the passive avoidance task requires two waves of glycoprotein synthesis, the first occurring immediately after training and the second 5–8 h later, the present results suggest strongly that isoforms of N-CAM molecules with a low level of sialic acid are involved specifically in the establishment of an enduring memory for the experience of the passive avoidance task in chicks, possibly by stabilising changes in synaptic connectivity that encode the memory.